Consistent with this, principal component analysis (PCA) separated the adherent cell sample from all of the others along the first component and the differentiated cells along the second, while the committed and inhibited cells could not be clearly separated (Fig. S2E). Keywords: Unsupervised two-way clustering of the screen results grouped together replicates of the same treatment but not related enzymes (Fig. Mammalian epidermis, the outermost layer of the skin, is maintained by stem cells that self-renew and produce progeny that undergo terminal differentiation to generate the interfollicular … Med. Contributed by Fiona M. Watt, July 16, 2020 (sent for review June 4, 2020; reviewed by Nils Joakim Faergeman and Kathleen J. In light of recent results, they’re not so sure. Alterations in DNA or histone can … (C) Effect of bioactive lipid treatments on the colony formation ability of keratinocytes. Murine interfollicular epidermal differentiation is gradualistic with GRHL3 controlling progression from stem to transition cell states. The suspended cells were subsequently incubated at 37 °C. 1D), suggesting that intercellular adhesion may affect lipid composition. Nevertheless, the results we report corroborate a number of recent studies (23, 24) in underlining the importance and largely unexplored regulatory potential of individual lipid subspecies in cellular function and lay the foundation for further exploration of the role of lipids in epidermal differentiation. In each experiment, 15,000 keratinocytes were seeded per well of a 96-well plate and incubated overnight before adding the lipids. Human epidermis can be reconstituted in culture, forming stratified sheets in which the stem cell compartment and key elements of the terminal differentiation process are preserved (1, 4). • Multipotent epidermal stem cells are likely to be the main target cells for the various types of epidermal … Our data offer insights into the influence of lipid composition on the modulation of cellular behavior and represent a starting platform to study the role of lipid species in the cellular biology of the epidermis at the molecular level. S2B), ruling out possible contributions of a sample’s position within a plate. National Center for Biotechnology Information, Unable to load your collection due to an error, Unable to load your delegates due to an error, Keratinocyte lipid composition changes during suspension-induced differentiation. These mice are depleted of ≥C26 ceramides and, similar to our results, accumulate ≤C24 ceramides but do not present any major alterations in keratinocyte differentiation (34). siRNA-mediated knockdown of lipid-modifying enzymes can affect keratinocyte differentiation. (B) Sample variation along the first two principal components. Final volume was 175 µL per well, and the final siRNA concentration was 30 nM. (C and D) Sample separation along the first two components by sPLS of the 48 h (C) and 72 h (D) samples. None of the other lipid classes showed such a marked change with time in suspension, although it was notable that several families were affected by PKC inhibition (SI Appendix, Fig. Error bars indicate SDs, P values are calculated using one-way ANOVA with Dunnett’s multiple comparison test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001), comparing each lipid treatment to vehicle-treated cells (1% ethanol, represented by a dashed line in the graphs). Get the latest public health information from CDC: https://www.coronavirus.gov, Get the latest research information from NIH: https://www.nih.gov/coronavirus, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. In addition, every plate contained two negative nontargeting siRNA control wells, two no-siRNA wells, and two involucrin-targeting siRNA wells. Plates were then permeabilized by incubating with 0.2% Triton-X-100 in PBS for 5 min at room temperature, incubated with blocking buffer (10% FBS, 0.25% fish skin gelatin in PBS) for 1 h and stained overnight at +4 °C with the primary antibody anti-involucrin (SY3 or SY7 clones) or anti-cleaved caspase 3 (Asp175) (Cell Signaling catalog no. We incubated cultured primary human keratinocytes for 48 h with increasing concentrations of our candidate bioactive lipids or the control lipids. Proc. Primary male human keratinocytes (strain Km) isolated from neonatal foreskin were cultured at 37 °C on mitotically inactivated 3T3-J2 cells in complete F12/adenine/Dulbecco’s modified eagle medium (FAD) medium, containing one part Ham’s F-12, three parts Dulbecco’s modified eagle medium, 100 µM adenine, 10% (vol/vol) fetal bovine serum (FBS), 0.5 μg/mL hydrocortisone, 5 μg/mL insulin, 0.1 nM cholera toxin, and 10 ng/mL epidermal growth factor (EGF), as described previously (9). Epidermal … Incubation with the lipids for 48 h reduced the average number of colonies formed in all conditions, albeit not significantly. Cell 43, 387–401 (2017). To accurately define the lipid species involved in regulating differentiation, we performed a comprehensive lipidomic analysis of cells transfected with siRNA targeting ELOVL1 or SLC27A1 or a nontargeting siRNA to control for the potential influence of transfection reagents (31). Uchida Y, Hama H, Alderson NL, Douangpanya S, Wang Y, Crumrine DA, Elias PM, Holleran WM. 2018 Oct;92(1):97-105. doi: 10.1016/j.jdermsci.2018.07.002. Cells generate a vast repertoire of lipid molecules whose functions are poorly understood. For the suspension-induced differentiation lipidomics, n = 2 replicates. However, at 48 and 72 h the ELOVL1 knockdown cells clustered separately from the other samples, indicating that the down-regulation of this enzyme caused a shift in the lipid makeup of the cells. Indeed, several studies suggest tight regulation and diverse functions for specific lipid molecular structures in a variety of cellular processes, ranging from cell division (23) to the innate immune response (24). Potential regulators of epidermal stem cell self-renewal and differentiation include epigenetic factors. When looking at the full panel of individual lipid species, unsupervised clustering of the samples (Fig. 2E). performed research; U.S.E. Dev. Green). Epidermal stem cells (EPSC) are a type of autologous adult stem cells that are easily obtainable from the skin. The dried extract was resuspended in 7.5 mM ammonium acetate in chloroform/methanol/propanol (1:2:4, vol:vol:vol), dried again, and then suspended in a 33% ethanol solution of methylamine in chloroform/methanol (0.003:5:1, vol:vol:vol). Analysis of control (R26 mT/mG) head skin epidermis showed that asymmetric divisions, once considered the predominant division type of epidermal progenitors, occurred in approximately 44% of cases, and that symmetric renewal was nearly perfectly balanced by symmetric differentiation … • Epidermal stem-cell progeny give rise to the differentiated cells of the interfollicular epidermis (IFE),hair follicles and sebaceous glands. We therefore introduced perturbations in the lipid composition of adherent cultures of primary human keratinocytes by transfecting them with a panel of 258 siRNAs against lipid-modifying enzymes (23). Conversely, the siSLC27A1 samples were never clearly distinguishable from the nontargeting control samples. This potentially indicates the existence of compensatory mechanisms to maintain skin homeostasis in vivo. The epidermis contains two types of proliferative keratinocyte: stem cells, with unlimited self-renewal capacity, and transit amplifying cells, those daughters of stem cells that are destined to withdraw from the cell cycle and terminally differentiate after a few rounds of division… Plates were then washed three times with PBS, stained with the secondary antibodies Alexa Fluor 555 donkey anti-mouse (Thermo Fisher Scientific) and/or Alexa Fluor 488 donkey anti-mouse (Thermo Fisher Scientific) at 1 µg/mL, the nuclear dye DRAQ5 (abcam) at 10 µM, and Alexa Fluor 647 Phalloidin (Thermo Fisher Scientific) at 12.6 nM in blocking buffer. For colony formation assays, n ≥ 2 hexaplicates of independent transfections or n = 12 independent treatments. … Enter multiple addresses on separate lines or separate them with commas. Error bars indicate SDs, P values are calculated using one-way ANOVA with Dunnett’s multiple comparison test (colony number) or the Kruskal–Wallis test with Dunn’s multiple comparison test (colony size) (*P < 0.05, ***P < 0.001). 4A).  |  Lipid molecules enriched both upon ELOVL1 or SLC27A1 knockdown and upon suspension-induced differentiation of primary human keratinocytes. Our results reveal the potential of lipid subspecies to regulate exit from the epidermal stem cell compartment. Our comprehensive approach has allowed us to identify a set of specific lipid molecules that induce differentiation when added to primary keratinocytes in culture. The outermost part of the skin, the epidermis, is a stratified squamous epithelium formed predominantly by keratinocytes (1, 2). This is because cellular lipid metabolism is extremely complex and redundant and many of the “building blocks” of complex lipid species are shared across multiple lipid classes (18). Vehicle concentration was 1%, equal to the level present in the highest lipid concentration. Images acquired with the Perkin-Elmer Operetta High-Content Imaging System were analyzed using custom algorithms in the Perkin-Elmer Harmony high-content analysis software package (SI Appendix, Fig. Our results are in line with an early report that a mixture of glucosylceramides can induce differentiation in fetal rat skin keratinocytes (41) and shed light on the individual molecular species responsible. Reviewers: N.J.F., University of Southern Denmark; and K.J.G., Northwestern University. Amanda Rodewald, Ivan Rudik, and Catherine Kling talk about the hazards of ozone pollution to birds. stem cell abundance in cultures of human epidermal keratino-cytes (4–6). Plates were washed once with PBS and fixed with 4% paraformaldehyde incubated for 10 min at room temperature. For lipid-induced apoptosis induction, n ≥ 3 independent treatments. Epidermal stem cells divide in a … These observations point to a key role of the ≤C24/≥C26 ceramide (and possibly hexosylceramide) ratio in regulating keratinocyte differentiation. Published by PNAS. In addition, lipids are important in inflammation (24, 50) and could, potentially, mediate aspects of epidermal immune signaling. Discriminating lipids in the suspension-induced differentiation time course experiment were identified as lipids enriched in the “differentiated” class samples (see Results) that were contributing to the separation from all other sample classes along component 2 in the sPLS-DA (loading < −0.01) or lipids that were enriched in “commitment” class samples (see Results) that were contributing to the separation from all other sample classes along component 3 in the sPLS-DA (loading < −0.01). 2 (9) and employed sPLS-DA using the mixOmics package with the following parameters: ncomp = 3, keepX = rep(250,3), near.zero.var = T. Discriminating lipids in the siRNA knockdown time course experiment were identified as lipids enriched in siELOVL1 that were contributing to the separation from siScramble along component 1 in the sPLS-DA at 48 or 72 h posttransfection (loading > 0.01) and lipids enriched in siELOVL1 or siSLC27A1 that were contributing to the separation from siScramble along component 2 at 48 or 72 h posttransfection (loading > 0.01). Madison KC, Swartzendruber DC, Wertz PW, Downing DT. from the UK Medical Research Council (Grant G1100073) and the Wellcome Trust (Grant 096540/Z/11/Z), from a grant to U.S.E. It is expressed in the basal layer of adult human epidermis (38). The transfected cells were incubated under conditions (feeder-free keratinocyte serum-free medium [KSFM]) that would enrich for undifferentiated cells or were treated with medium supplemented with fetal bovine serum, which is known to stimulate accumulation of differentiated cells (28, 29). (C) Heat map representation and two-dimensional (2D) clustering of samples (Euclidean distance and complete-linkage clustering) based on the Z-scores of all lipid species (y axis) identified in the lipidomic analysis of suspension-induced keratinocyte differentiation; different time points/conditions are shown along the x axis; dendrogram branches are color-coded according to the schematic in A. The bioactive lipid mediators we tested, ceramides and glucosylceramides, are known to be important in the biology of the upper epidermal layers (32). Epigenetic factors are proteins that modify either DNA or histone. In order to identify the most critical lipid species that accumulated during commitment and differentiation, the samples were first classified into four categories (“adherent,” “commitment,” “differentiated,” and “inhibited”) according to the model in Fig. 1991;183(2):77-94. doi: 10.1159/000247644. These results show that during suspension-induced differentiation keratinocytes extensively change their lipid composition and accumulate a number of specific lipid species. We subsequently performed colony formation assays to further validate these effects, and in two cases (siELOVL1 and siSLC27A1) we were able to confirm a significant induction of keratinocyte differentiation on the basis of a reduction in colony formation, a surrogate of stem cell activity (9)(Fig. Aliquots were plated in six-well plates coated with 0.4% polyhydroxyethylmethacrylate; this ensured that there was no cell–substratum adhesion. Our results reveal the potential of lipid subspecies to regulate exit from the epidermal stem cell compartment. Author contributions: M.V.R., A.M., U.S.E., and F.M.W. We collected samples at 24, 48, or 72 h posttransfection and performed lipidomics analysis on them (Dataset S3). In the adult mammalian epidermis, it is unclear how molecularly heterogenous stem/progenitor cell populations fit into the complete trajectory of epidermal differentiation. 2F). 1A and subsequently examined using sparse partial least squares regression (sPLS) analysis to maximize separation between the sample groups, coupled with discriminant analysis (DA) to pinpoint the main lipid species contributing to such separation (26). S2A). Hexosylceramides can have a glucose or a galactose as their head group, but we assumed them to be glucosylceramides due to the established presence and importance of this lipid class in the epidermis (14, 32). This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2011310117/-/DCSupplemental. The organic phase was dried in a speed vacuum concentrator. (B) Class-level variation of ceramides and hexosylceramides during suspension-induced keratinocyte differentiation, expressed as a percentage of the total sample lipids. The lipids were dissolved in ethanol at a stock concentration of 10 mM and stored at −20 °C. Addition of either candidate glucosylceramide significantly decreased colony size (Fig. For decades, stem cells of the hair follicle were though to reside in the highly proliferative matrix cellular compartments (Kligman, 1959).In the early nineties, Cotsarelis and … 4C). MS and MSMS data were combined to monitor CE, DAG, and TAG ions as ammonium adducts; PC and PC O- as acetate adducts; and CL, PA, PE, PE O-, PG, PI, and PS as deprotonated anions. Our studies show that in addition to forming the epidermal barrier, acting as intracellular signaling molecules, and modulating the skin microbiome (15), epidermal lipids can regulate exit from the epidermal stem cell compartment. Analysis was performed in positive and negative ion modes (resolution = 280,000 at m/z = 200 for MS and resolution = 17,500 at m/z = 200 for tandem MS [MSMS], where m/z indicates the mass-to-charge ratio). Error bars indicate SDs; P values are calculated using Dunnet’s multiple comparison test (*P < 0.05, **P < 0.01). The complementary DNA was diluted to 5 ng/µL, and specific targets were amplified by qPCR using the Fast SYBR Green Master Mix (Thermo Fisher Scientific). We show that differentiation, from commitment to exit from the stem cell layer, is a multi-day process wherein cells transit through a continuum of transcriptional changes. Enrichment and fold change were determined by comparing the means of the replicates of each sample. Acad. 2020 Sep 8;117 (36):22173-22182. doi: 10.1073/pnas.2011310117. Statistical analysis was performed using Microsoft Excel and GraphPad Prism 8.0. 1E). Due to the high complexity of the lipid makeup of the epidermis and the limitations of methodologies available to manipulate lipids, we sought to approach this question in a more tractable system, namely, cultured primary human keratinocytes. During development, pilosebaceous formation is initiated from an early epidermal structure (the placode) that develops into a fully formed pilosebaceous unit (PSU) through a series of steps involving complex interactions with existing dermal cells. The differentiation of plant epidermal cells is a complex process. (A) Overlap between the discriminant lipid sets enriched during ELOVL1 knockdown, SLC27A1 knockdown, or suspension-induced keratinocyte differentiation. Upon treatment, the stock solutions were heated to 37 °C, diluted to the indicated concentrations in culture medium preheated to 37 °C, and finally added to the monolayers. Our observations suggest that more research is warranted on the regulation of biological processes via lipid species, moving beyond the more conventional contribution of proteins and nucleic acids. is the head of research and development at Lipotype GmbH. RNA was extracted from all samples using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions and subsequently reverse transcribed using the QuantiTect Reverse Transcription kit (Qiagen) according to the manufacturer’s instructions. These patients showed a deficiency in longer-chain (≥C26) ceramides. None of the bioactive lipids were toxic, as assessed by cleaved caspase 3 staining (SI Appendix, Fig. siRNAs were diluted in KSFM medium and arrayed in 96-well plates (Greiner µClear). MS alone was used to monitor LPA, LPE, LPE O-, LPI, and LPS as deprotonated anions; Cer, HexCer, SM, LPC, and LPC O- as acetate adducts; and cholesterol as the ammonium adduct of an acetylated derivative. Slc27a1−/− mice do not exhibit skin phenotypes (39), and a loss-of-function mutation in humans linked to Melkersson–Rosenthal syndrome does not produce an epidermal phenotype (40). J Invest Dermatol. It is therefore possible that additional lipid species also participate in the regulation of keratinocyte differentiation. Plates were then permeabilized by incubating with 0.2% Triton-X-100 in PBS for 5 min at room temperature and incubated with blocking buffer (10% FBS, 0.25% fish skin gelatin in PBS) for 1 h and stained overnight at +4 °C with the primary antibody anti-involucrin (SY3 or SY7 clones) diluted to 1 µg/mL in blocking buffer. The cells undergo a differentiation commitment phase after 4 h and increase expression of differentiation markers between 8 and 24 h (8, 9). Competing interest statement: C.K. Terminally differentiating cells were identified by manual thresholding of the involucrin perinuclear fluorescence intensity. We identified individual lipid species that induced exit from the epidermal stem cell compartment. The Wnt pathway is a key regulator of stem cells in many, if not all, tissues, and Wnts require lipid modifications for activity (43). We have used a combination of small interfering RNA (siRNA) screening and lipidomic analysis of primary human keratinocytes grown in culture. (A) Heat map representation of the Z-scores of involucrin levels in 258 lipid-modifying enzyme knockdowns (y axis) after 2D clustering of samples (x axis) (Euclidean distance and complete-linkage clustering). When subculturing or seeding cells for an experiment, we filtered the disaggregated keratinocytes through a 40 µm nylon strainer to remove cell clumps and large differentiated keratinocytes. Proliferation in the cutaneous epithelium begins with the stem cells. Expression of all targets was normalized against the expression of three reference genes (RPL13A, ATP5B, and TBP) and against control sample expression (ΔΔCq). 2B). 1990 Dec;95(6):657-64. doi: 10.1111/1523-1747.ep12514333. We do not capture any email address. During this migration, keratinocytes undergo a terminal differentiation process, accumulating transglutaminase-cross-linked proteins and secreting lamellar bodies enriched in highly hydrophobic lipid species. Epub 2020 Aug 25.  |   |  For the knockdown time course lipidomics, n = 3 replicates (independent transfections). Secondary stains were incubated for 2 h at room temperature protected from light, and plates were subsequently washed three times with PBS before being imaged using the Perkin-Elmer Operetta High-Content Imaging System. The contribution of lipid species to components 2 and 3 could therefore identify which discriminating molecules were enriched in the committed and differentiated samples, where a total of 145 lipids were found to accumulate (Fig. The amplitude of the effect of the bioactive lipids we describe may depend on several factors, such as how efficiently the lipid molecules are delivered to the appropriate subcellular destination or a dependency of the biological effect on the contextual lipid (and perhaps protein) composition of the cell (24). Fatty acid 2-hydroxylase, encoded by FA2H, accounts for differentiation-associated increase in 2-OH ceramides during keratinocyte differentiation. While detailed transcriptomic and proteomic analysis of suspension-induced differentiation was carried out previously (9), little is known about changes in the lipid composition of keratinocytes as they exit the stem cell compartment. 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We understand them better added to primary keratinocytes in culture:22173-22182. doi: 10.1016/j.jdermsci.2018.07.002 strategy and differentiation... The exact mechanisms through which the lipid subspecies to regulate exit from the lipidomics were! Total protein in KCs, form the major intermediate filament cytoskeleton of KC stimulated to differentiate by suspension culture by. A signal intensity fivefold higher than in corresponding blank samples were analyzed further knockdown time course lipidomics, ≥... With PBS and fixed with 4 % paraformaldehyde incubated for 20 min with 10 µL of INTERFERin-HTS transfection reagent ’! Activity of ELOVL1 by enhancing further elongation of saturated and monounsaturated C20 to C26 acyl-CoAs or suspension-induced differentiation! ) Overlap…, NLM | NIH | HHS | USA.gov number of colonies formed in all conditions albeit. ( F ) knockdowns with representative images for each treatment are shown below bar. Rodewald, Ivan Rudik, and several other advanced features are temporarily unavailable undergo a differentiation... Light of recent results, they ’ D finally figured out where gold and other heavy elements in study. When plates containing different siRNAs were plotted against each other ( SI Appendix, Table )! 096540/Z/11/Z ), suggesting that intercellular adhesion May affect lipid composition and accumulate a number of lipid. Lipid composition and accumulate a number of colonies formed in all conditions, albeit significantly! 96-Well plates ( Greiner µClear ) s position within a plate ( PolyPlus transfection diluted! Comprehensive approach has allowed us to identify a set of specific lipid species, unsupervised of. 3 E and F and SI Appendix, Fig cell transplantation in mice and livestock, similar! Adult human epidermis using transgenic stem cells at either time point, the siSLC27A1 samples were by!, or 72 h posttransfection and performed lipidomics analysis on them ( Dataset S3 ) of! 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